The β-cells of the pancreatic islets secrete a single chain precursor of insulin, known as pro-insulin which upon proteolysis results in the biologically active polypeptide insulin. The insulin molecule is a highly conserved across species and generally consists of two chains of amino acids linked by disulfide bonds. The natural human insulin molecule (mw 5,807 Daltons), has A-chain of 21 amino acid residues with glycine at the amino terminus; and a B-chain of 30 amino acid residues with phenylalanine at the amino terminus. Insulin may exist as a monomer or may aggregate into a dimer or a hexamer formed from three of the dimers. The monomer has the ability to bind to receptors and is the biologically active form.
Insulin polypeptide is the primary hormone responsible for controlling the transport, utilization and storage of glucose in the body. A defect in the carbohydrate metabolism as a result of insufficient production of insulin or reduced sensitivity of the receptor to insulin leads to the biological disorder diabetes. Diabetes impairs the normal ability to use glucose as a result increases blood sugar levels (hyperglycemia). As glucose accumulates in the blood, excess levels of sugar are excreted in the urine (glycosuria). Other symptoms of diabetes include increased urinary volume and frequency, thirst, itching, hunger, weight loss, and weakness. Diabetes when left untreated leads to ketosis, followed by acidosis with nausea and vomiting. As the toxic products continue to build up, the patient goes into a diabetic coma, which leads to the patient's death. There are two types of diabetes Type I is insulin-dependent diabetes mellitus, or IDDM. IDDM was formerly referred to as “juvenile onset diabetes.” In IDDM, insulin is not secreted by the pancreas and must be provided from an external source. Type II or adult-onset diabetes can ordinarily be controlled by diet, although in some advanced cases insulin is required.
Traditionally bovine and porcine insulin were used almost exclusively to treat diabetes in humans. With the development of recombinant technology commercial scale manufacture of human insulin was made possible by fermentation. Furthermore, genetically engineered insulin analogs having biological activity comparable to that of natural human insulin were developed to combat the disease. However, treatment of diabetes typically requires regular injections of insulin. Due to the inconvenience of insulin injections, various approaches have been attempted to formulate insulin for administration by non-injectable routes. A list of such publications include: U.S. Pat. No. 4,338,306 (Kitao et al.) reports a pharmaceutical compositions of insulin and fatty acids having 8 to 14 carbon atoms and nontoxic salts thereof for rectal administration of insulin; U.S. Pat. No. 4,579,730 (Kidron et al) reports an enterocoated insulin compositions with a bile acid or alkali metal salt thereof for the oral administration of insulin; U.S. Pat. No. 5,283,236 (Chiou et al.) reports an insulin composition with a permeation-enhancing agent to aid systemic absorption of higher molecular weight polypeptides, as well as peptidase inhibitors for systemic delivery of insulin through the eyes wherein the drug passes into the nasolacrimal duct and becomes absorbed into circulation; U.S. Pat. No. 5,658,878 (Backstrom et al.) reports an insulin and sodium salt of a saturated fatty acid of carbon chain length 10 (i.e., sodium caprate), 12 (sodium laurate), or 14 (sodium myristate) which enhances the absorption of insulin in the lower respiratory tract; U.S. Pat. No. 5,853,748 (New et al.)—reports an enteric-coated composition of insulin, a bile salt or bile acid, and carbonate or bicarbonate ions, used to adjust the pH of the gut to a pH of from 7.5 to 9 for the oral administration of insulin. U.S. Pat. No. 6,200,602 (Watts et al) reports a drug delivery composition of insulin for colonic delivery of insulin with an absorption promoter which includes a mixture of fatty acids having 6 to 16 carbon atoms and its salts or a mixture of mono/diglycerides of medium chain fatty acids along with a dispersing agent, in a coating to prevent the release of the insulin and absorption promoter until the tablet, capsule or pellet reaches the proximal colon.
Several attempts to deliver insulin by oral administration are found in literature. The problems associated with oral administration of insulin to achieve euglycemia in diabetic patients are well documented in pharmaceutical and medical literature. Digestive enzymes in the GI tract rapidly degrade insulin, resulting in biologically breakdown products. In the stomach, for example, orally administered insulin undergoes enzymatic proteolysis and acidic degradation. Survival in the intestine is hindered by excessive proteolysis. In the lumen, insulin is barraged by a variety of enzymes including gastric and pancreatic enzymes, exo- and endopeptidases, and brush border peptidases. Even if insulin survives this enzymatic attack, the biological barriers that must be traversed before insulin can reach its receptors in vivo may limit oral administration of insulin. For example, insulin may possess low membrane permeability, limiting its ability to pass from the lumen into the bloodstream.
Pharmaceutically active polypeptides such as insulin have been conjugated with polydispersed mixtures of polyethylene glycol or polydispersed mixtures of polyethylene glycol containing polymers to provide polydispersed mixtures of drug-oligomer conjugates; U.S. Pat. No. 4,179,337 (Davis et al) reports conjugating polypeptides such as insulin with various polyethylene glycols such as MPEG-1900 and MPEG-5000 supplied by Union Carbide. U.S. Pat. No. 5,567,422 (Greenwald) reports the conjugation of biologically active nucleophiles with polyethylene glycols such as m-PEG-OH (Union Carbide), which has a number average molecular weight of 5,000 Daltons.
Conjugation of polypeptides such as insulin with polyethylene glycol modified glycolipid polymers and polyethylene glycol modified fatty acid polymers are reported in U.S. Pat. No. 5,359,030 (Ekwuribe et al.).
U.S. Pat. No. 6,011,008 (Domb et al.) reports a method for producing a water-soluble polysaccharide conjugate of an oxidation-sensitive substance comprising activating the polysaccharide to a dialdehyde by periodate oxidation; (b) purifying the dialdehyde from interfering anions and by-products; and (c) coupling the substance to the purified dialdehyde by Schiff base formation to form the conjugate. Optionally, the conjugate of step (c) is reduced to an amine conjugate by a reducing substance. Insulin was conjugated to oxidized AG (arabinogalactan) via an amine or imine bond by reacting a solution of pure oxidized AG (arabinogalactan) in borate buffer solution at pH 8.9 with insulin at 4° C. overnight. The clear solution was dialyzed through a cellulose dialysis and the solution was lyophilized to yield 115 mg of a white solid.
U.S. Pat. No. 6,022,524 (Maisano et al.) reported conjugation of Gd-DTPA with porcine insulin in a solution of DTPA and dimethylsulfoxide (DMSO) which was prepared by heating and stirring, then cooled at room temperature and added with a solution of 11.73 g NHS (0.102 mol) in 300 ml DMSO, then, drop by drop, with a solution of 19.6 g of N,N′-dicyclohexylcarbodiimide (0.097 mol) in 400 ml DMSO. The mixture is stirred for 16 hours, then filtered and the filtrate is concentrated by evaporation at 50.degree. C. and 5 Pa to a thick oil of an about 160 ml volume.
U.S. Pat. No. 6,309,633 (Ekwuribe et al.)—reports the use of solid insulin for conjugation of insulin with laurate PEG5 in presence of Triethylamine and DMSO at room temperature. The reaction was monitored via HPLC every 30 mins. The conjugate was purified using a preparative HPLC.
U.S. Pat. No. 6,828,297 (Ekwuribe et al.) reports methods for making PEG7-Hexyl-Insulin by using zinc or zinc free human insulin for conjugation with activated oligomer and purification of B29 modified PEG7-Hexyl-Insulin, insulin in dimethylsulfoxide and triethyl amine was reacted with activated oligomer at 22+/−4° C. The crude reaction mixture is dialyzed or difiltered to remove organic solvents and small molecular weight impurities, exchanged against ammonium acetate buffer and lyophilized; which is further subjected to RP-HPLC equilibrated with 0.5% triethylamine/0.5% phosphoric acid buffer (TEAP A). The column was eluted with a gradient flow using TEAP A and TEAP B (80% acetonitrile and 20% TEAP A) solvent system. Fractions containing the conjugate were pooled and the elution buffer and solvent were removed by dialysis or diafiltration against ammonium acetate buffer and lyophilized to produce white powder of PEG7-hexyl-insulin, B29 monoconjugate (purity>97%). Currently, the existing prior art teaches use of pure insulin powder or crystals as the starting material for making conjugated insulin wherein the insulin used is a biologically active form.
The instant invention facilitates the making of insulin-oligomer conjugate from the precursor IP-X of formula Z-[B-Chain]-Q-[A-Chain], where Z is a leader where Z is a leader peptide sequence, B-Chain is B chain of human insulin or its analog, Q is a linker peptide sequence between the A and B chain, A-chain is the A chain of human insulin or its analog.
The precursor IP-X is conjugated with an oligomer at the LysB29 position on the B-chain and the N-terminus amino acid of the leader peptide Z. The IP-X-oligomer conjugate is then subjected to protease treatment followed by purification to get an active insulin-oligomer conjugate.
The starting material is the fermented broth containing the precursor IP-X. The broth containing the IP-X is subjected to a combination of chromatography like ion-exchange, HPLC, RP-HPLC and crystallization to purify the IP-X.
The instant invention is a more simplified and economical in the making of an insulin-oligomer conjugate wherein several steps involved in obtaining pure insulin crystals in biologically active form are circumvented e.g, transpeptidation of the insulin precursor and cleaving the insulin precursor to get the active insulin and several chromatographic purification steps e.g., ion-exchange chromatography, HPLC and RP-HPLC to get the pure insulin crystals.